1a. Objectives (from AD-416)
Objective 1: Spatially characterize conditions within manure-impacted environments that lead to pathogen persistence, nutrient losses, and gaseous emission hot spots.
Sub objective 1A: Develop, evaluate, and validate high-throughput methods for the detection, quantification and identification of microbial pathogens and fecal indicator organisms from environmental sources such as feedlot runoff, and ground and surface water samples.
Sub objective 1B: Identify areas within cattle feedlot pens prone to gas emissions (odors, nutrients, and greenhouse gases) and evaluate the relationship between those areas and the spatial distribution of pathogens and fecal indicator microorganisms.
Sub objective 1C: Determine the spatial distribution of contaminants, nitrogen-transforming activities, and microorganisms responsible for nitrogen transformation in a contamination plume originating from a cattle feedlot runoff holding pond.
Objective 2: Develop land application practices that incorporate the use of manure as a nutrient source for crop production while minimizing potential adverse environmental impacts.
Sub objective 2A: Utilize rainfall simulations tests to evaluate the potential for pathogen and nutrient runoff from manure applied to cropland.
Sub objective 2B: Utilize laboratory soil columns to evaluate odor compound emissions after manure application to cropland.
Objective 3: Develop manure management practices (e.g. vegetative treatment systems) to control gaseous emissions, nutrients, and microorganisms (e.g. E. coli O157:H7).
Sub objective 3A: Determine the movement and persistence of nutrients, pathogens, and fecal indicator organisms within the vegetative treatment system (VTS) during standard and non-standard VTS operation.
Sub objective 3B: Evaluate the antibiotic resistant phenotypes and genotypes of bacteria isolated directly from manure, and compare to bacteria from the corresponding feedlot runoff, and the agricultural fields to which the manure has been applied.

1b. Approach (from AD-416)
Experiments will be conducted in the field and in the laboratory to evaluate gas emissions, nutrient transport, and microbial transport and fate associated with specific types of confined animal feeding operations and wastewater treatment processes. New high-throughput methods for the detection of specific fecal microorganisms and antibiotic resistance genes will be developed and applied towards this goal. In field studies, specific areas within manure-impacted environments (beef cattle feedlot pens, vegetative treatment areas for feedlot runoff treatment, a cattle feedlot holding pond groundwater contamination plume, and crop fields utilizing manure as fertilizer) will be identified that disproportionately emit gases (odor compounds, ammonia, and greenhouse gases) or have a large potential for nutrient or pathogen transport through the use of flux chambers and gas chromatography, by the use of artificial rainfall simulators, and by microbiological methods. In laboratory studies, molecular techniques will be used to assess microbial communities in order to understand the relationships between microbes and environmental processes affecting nutrients, emissions, and pathogen fate.

3. Progress Report
During the first year of the new research project we made progress on all objectives and met all milestones. For objective #1, quantitative methods for the detection of three Shiga-toxin producing Escherichia coli (STEC) serotypes were evaluated in our laboratory using both research and field samples. We are now routinely using the quantitative STEC O157 assay in other projects, and the validation work will be written up for publication in a peer reviewed journal. Methods for detection and enumeration of STEC O26 and O111 methods were also evaluated but were found to be resource and labor intensive. Application of these methods were used last year to isolate the source of an STEC O111 outbreak in a Colorado prison (source was a dairy). These techniques are now used in multiple projects to evaluate the fate and transport of STEC O157, O111, and O26 in runoff from manure-amended fields.
For objective #2, a series of manure application studies on cropland were conducted and information on the potential runoff of nutrients, pharmaceutically active compounds, antibiotic resistance genes, and pathogens was collected. The effects of cover crops and wheat strips on nutrient and pathogen losses were determined. A complementary study in the laboratory was also conducted using swine manures applied to soil columns and followed the emission of various odor compounds over time. A consistent pattern was observed with volatile fatty acids emitted immediately for a shorter period of time and aromatic compounds emitted over a longer time frame.
Although no milestones were scheduled for this year in objective #3, initial work at the vegetative treatment area (VTA) was conducted examining pharmaceutically active compounds, pathogens, and nutrient dynamics when cattle feedlot runoff was applied. An initial set of deep cores was also collected to serve as a benchmark for the movement of runoff microbes and compounds into the soil. Initial results indicate the VTA was able to utilize nutrients and remove microbes applied to the treatment areas.

4. Accomplishments
1. Animal diet affects manure-borne pathogen transport. Animal diet can affect the physical, chemical, and microbiological composition of the resulting manure. For example, the feeding of distiller’s grains as part of a finishing diet for cattle is associated with higher numbers of the pathogenic E. coli O157:H7. Little is known, however, about the impacts of diet on the transport of manure-borne microorganisms once manure is land-applied. ARS researchers in Lincoln, NE, investigated bacterial and parasitic bacterial virus transport in runoff from fields amended with manure from animals fed either corn or 40% wet distiller’s grain. They determined that diet significantly affects parasitic bacterial viruses, but not bacterial transport from manure-amended fields. This information is being used to develop land application practices that incorporate the use of manure as a nutrient source for crop production while minimizing potential adverse environmental impacts.

2. Cattle feedlot pens are more than just uncomposted manure. The public, and much of the food safety community, tend to think of Escherichia coli O157:H7 as being synonymous with cattle feces, and the feedlot pen that collects the feces is conceptualized as being just packed uncomposted manure. Using DNA-based bacterial community profiling, ARS researchers in Lincoln, NE demonstrate that the bacterial communities in the feedlot pen are distinct from those of the fecal source material. Fecal microorganisms face a challenging environment on the pen surface compared to the animal gastrointestinal tract that selects for the persistence of some microorganisms over others. A better understanding these environmental challenges facing fecal pathogens like E. coli O157:H7 can lead to new ways of controlling E. coli O157:H7.